JP2916947B2 - Method for stabilizing CPB-I and pharmaceutical composition - Google Patents

Description

The present invention relates to a method for stabilizing CPB-I or recombinant CPB-I which is useful as an anticoagulant or a therapeutic agent for skin / corneal diseases, and a pharmaceutical composition. The separation and purification of these,
The present invention relates to a stabilization method useful for performing operations such as freeze-drying and a pharmaceutical composition excellent in storage safety.

[Problems to be solved by conventional technology and invention]

CPB-I is widely distributed in in vivo tissues and secretions, including human placenta (Chem. Pharma. Bull, 38, 1957-196).
0,1990), and a substance having a physiological activity such as an anticoagulant effect existing in the cytoplasm within a cell.

CPB-I can be extracted from human or animal organs (JP-A-62-174023). The CPB-I thus obtained has the following properties.

Molecular weight (SDS-polyacrylamide gel electrophoresis, reduced state) 34,000 ± 2,000 Isoelectric point (isoelectric point column electrophoresis using ampholite) 4.7 ± 0.1 Stability (a) Loss by heating at 50 ° C for 30 minutes Active (b) Stable at pH 4-10 (c) Stable at 37 ° C, 30 minutes in plasma Effect on blood coagulation system (a) Extend calcium recoagulation time (b) Extend prothrombin time (c) Activated partial thromboplastin Extend time Amino acid analysis Amino acid analysis shows aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, 1 /
2 The presence of cystine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine and arginine is observed.

In addition, CPB-I is obtained by using a gene fragment encoding human or animal CPB-I,
It can be expressed in yeast and the like (JP-A-64-20095). CPB- thus obtained by genetic recombination
I (hereinafter referred to as "recombinant CPB-I")
Has the following amino acid sequence:

Recombinant CPB-I can also be produced using yeast other than E. coli. In this case, an amino acid sequence in which an acetyl group is bonded to alanine at the amino terminus is obtained.

Such CPB-I or recombinant CPB-I can be made into various dosage forms such as a solution, a gelling agent, an ointment, an ophthalmic solution, and an injection.

However, it is necessary to separate and purify, freeze-dry, etc. during the formulation process, and after the formulation as described above,
It has to be stored for a long period of time, and at this time, there is a drawback that the activity of the CPB-I is reduced due to polymerization or the like.

Therefore, a method for stabilizing CPB-I or recombinant CPB-I and a stable pharmaceutical composition have been desired.

[Means for solving the problem]

The present inventors have conducted intensive research in view of the above situation,
By adding a basic amino acid to CPB-I or recombinant CPB-I, these can be stabilized, and even if an operation such as separation / purification / freeze-drying is performed, it can be formulated and stored for a long time. Found that the activity did not decrease, and completed the present invention.

That is, the present invention relates to CPB-I or recombinant CPB-
CPB characterized by adding I and a basic amino acid
-I or a method for stabilizing recombinant CPB-I and CPB-
CPB-I or recombinant CPB, characterized by containing I or recombinant CPB-I and a basic amino acid
-I provides a stabilized pharmaceutical composition.

The basic amino acid used in the present invention includes, for example, lysine, arginine, ornithine and the like. In the case of preparation for injection, L-lysine or L-arginine is preferable. The amount of the basic amino acid is preferably about 0.6 to 20 mg per ml of a solution containing CPB-I or recombinant CPB-I (hereinafter referred to as “CPB-I etc.”). In addition, the amount of the stabilizer to be added to the powder,
What is necessary is just to make it the said solution density | concentration when the said powder is melt | dissolved. If the amount is less than 0.6 mg, it is difficult to achieve stabilization, and if the amount exceeds 20 mg, the effect cannot be expected to be significantly improved.

The method for adding the basic amino acid is not particularly limited, and CPB-
I, a method of directly adding a basic amino acid to water or a suitable buffer solution, and a method of mixing a basic amino acid powder with CPB-I or the like and adding / dissolving it in water, etc. What is necessary is just to select a method etc. suitably. The timing of addition is not particularly limited, and a separation and purification step of CPB-I or the like,
Any of the formulation steps may be used.

The stabilized composition of the present invention in which a basic amino acid is added to CPB-I or the like is preferably separated and purified, formulated or stored at 0 to 30 ° C, particularly 0 to 10 ° C in the case of a solution. When the solution is stored in a frozen state, the temperature is preferably 0 ° C. or lower, particularly -20 ° C. or lower.

Further, the composition of the present invention includes glucose, glucosamine, xylose, saccharose, dextran T-40,
Sugars such as mannitol, albumin and the like can be added.

The stabilized preparation composition containing CPB-I etc. of the present invention can be made into various dosage forms depending on the use. For example, when used as an anticoagulant, in the form of an aqueous injection or the like,
When used as a therapeutic agent for skin and corneal diseases, it can be in the form of a liquid, gel, cream, ointment, eye drop, ointment or the like.

〔The invention's effect〕

According to the method of the present invention, CPB-I and the like are frozen and thawed, separated and purified by dissolution in an aqueous medium, etc.
Activities such as BI are retained. In addition, the pharmaceutical composition of the present invention does not decrease the activity of CPB-I even when stored for a long time, and is useful as a pharmaceutical.

〔Example〕

Example 1 CPB-I 6.76 mg / dissolved in 10 mM phosphate buffer (pH 7.4)
As a stabilizer, aspartic acid, glycine, or lysine was added as a stabilizer to each of 5 mg / ml of a 100 μl solution.
Frozen at 0 ° C. and thawed at room temperature. After repeating this operation six times, analysis was performed by the following measurement method. Table 1 shows the results
Shown in

Determination of CPB-I polymer by HPLC 10 mM phosphate buffer (pH 7.
Tosoh HPLC gel filtration column TSK g equilibrated in 4)
20 μ of a sample solution diluted to 1 mg / ml in the same buffer as above was injected into el G3000SW _XL (7.8 × 300 mm). The polymer was separated at a flow rate of 1 ml / min, the absorbance of the sample eluted from the column was measured at a wavelength of 280 nm, and the ratio between CPB-I and the polymer was calculated from the area value.

Example 2 CPB-I 6.76 mg / dissolved in 10 mM phosphate buffer (pH 7.4)
L-lysine hydrochloride as a stabilizer in 0.04 ml / ml
100 μ of the solution containing 2020 mg was frozen at −40 ° C. and then thawed at room temperature. After repeating this operation five times, analysis was performed in the same manner as in Example 1. Table 2 shows the results.

Example 3 CPB-I 6.76 mg / dissolved in 10 mM phosphate buffer (pH 7.4)
10 mg / ml of L-lysine hydrochloride as a stabilizer
The anticoagulant activity of the solution to which was added was measured by the following method. Table 3 shows the results.

Measurement of anticoagulant activity 20 mM containing 0.5% bovine serum albumin and 0.15 M NaC1
100 μl of a sample solution diluted to 5 and 10 μg / ml with Tris-HCl buffer (pH 7.4) was added with 100 μl of lipoplastin 0.5% / ml from Mochida Pharmaceutical Co., Ltd. diluted with 30 mM CaCl ₂ solution.
Preincubated for minutes. In addition, saline at 37 ° C
200 μm of Ortho control plasma dissolved in 2 ml was added, and the time until the plasma coagulated was measured using a Coagtech TE-600 manufactured by Elma Optical.

──────────────────────────────────────────────────続 き Continuing from the front page (72) Inventor Satoshi Adachi 3190-202 Tsuyama, Kikuyo-cho, Kikuchi-gun, Kumamoto Pref. Int.Cl. ⁶ , DB name) C07K 14/47 A61K 38/17 A61L 47/18

Claims (4)

(57) [Claims] 1. A method for stabilizing CPB-I or recombinant CPB-I, comprising adding a basic amino acid to CPB-I or recombinant CPB-I. 2. A pharmaceutical composition comprising stabilized CPB-I or recombinant CPB-I, comprising CPB-I or recombinant CPB-I and a basic amino acid. 3. The method according to claim 1, wherein the basic amino acid is one or more selected from lysine, arginine and ornithine. 4. The pharmaceutical composition according to claim 2, wherein the basic amino acid is one or more selected from lysine, arginine and ornithine.